Wound healing is a complex process involving several cell types and growth factors for an effective closure. The normal wound healing process can be broadly classified into three stages namely the inflammatory, proliferative and maturation phases. The inflammatory phase lasts 0–2 days and involves an orderly recruitment of cells to the wound area. This is followed by the 2–6 day proliferative phase, in which fibroblasts, keratinocytes and other cells in the wound bed begin to actively proliferate to close the wound. The maturation phase follows the proliferative phase, peaking at 21 days, by which time the wound is completely healed by restructuring the initial scar tissue.
A problematic wound does not follow the normal time table for the healing process as described above. A problematic wound could fail to follow the normal healing process for any number of reasons, including nutrition, vascular status, metabolic factors, age, immune status, drug therapy, neurologic status and psychologic status, among others. Several local factors also play an important role in wound healing, including the presence of necrotic tissue in the area, infection, foreign body presence, degree of desiccation, presence of edema, pressure, friction, shear maceration and dermatitis.
It has been shown from wound fluid composition studies that growth factors play an important role in all three phases of wound healing. The cell types that are recruited to the wound area secrete growth factors that assist in and promote the wound healing process. Platelets, for example, are the first cell type to be recruited at the wound site, and initiate the wound healing process by secreting growth factors (i.e., platelet derived growth factors, or PDGF) which are chemotactic for other cell types. By so doing, the platelets assist in the recruitment and proliferation of additional cell types that promote synthesis of new tissue. In addition to the above mentioned functional properties, growth factors also have the ability to regulate protein synthesis within the cell and control intracellular signaling thus allowing cells to communicate with one another.
Since wound healing is a complex process which involves formation of connective tissue, and new blood vessels to nourish the site, it is evident that several growth factors come into play. In chronic wounds there is an increase in collagenase activity and higher levels of inflammatory cytokines. Additionally, there is an absence of growth factors in the wound fluid which causes the cells to be mitotically incompetent. All of these factors cause impaired wound healing. Some of these factors have been studied in the preclinical animal models as well as in the clinic. Most growth factor studies involving the wound healing process involve tests in the 20–25 day range, which appears to adequately model the normal wound healing process. However, it is now realized that to get 100% closure of problematic wounds, longer study periods such as long as 6 months or more would be advantageous.
The only FDA approved growth factor for wound healing use in the clinic is platelet derived growth factor (PDGF) marketed by Ortho-McNeil Pharmacuetical as REGRANEX®. REGRANEX® contains becaplermin, a recombinant human platelet-derived growth factor (rhPDGF-BB) for topical administration. Becaplermin is produced by recombinant DNA technology by insertion of the gene for the B chain of platelet derived growth factor (PDGF) into yeast. Becaplermin has a molecular weight of approximately 25 KD and is a homodimer composed of two identical polypeptide chains that are bound together by disulfide bonds. REGRANEX® is a non-sterile, low bioburden, preserved, sodium carboxymethylcellulose-based (CMC) topical gel, containing the active ingredient becaplermin and the inactive ingredients sodium chloride, sodium acetate trihydrate, glacial acetic acid, water for injection, and methylparaben, propylparaben, and m-cresol as preservatives and l-lysine hydrochloride as a stabilizer.
Studies of various growth factors in the wound healing process have been conducted. Some of the findings from these studies are summarized below:
1) PDGF-BB (the growth factor in REGRANEX®) is a chemoattractant for neutrophils, monocytes, and fibroblasts. In wound healing applications it has been shown to increase extracellular matrix deposition and enhance proliferation of fibroblasts. PDFG is not an angiogen, however. Thus, additional growth factors will be required for the healthy maintenance of neodermis.
2) Fibroblast Growth Factor (FGF) increases capillary density and proliferation of fibroblasts. A topical application in gel form was tested and it was shown that there was no systemic absorption of the protein (<1% of the dose detected).
3) Transforming growth factor β-2 (TGF β-2) is a growth factor that enhances proliferation of several cell types both in vitro and in vivo and has been tested in venous ulcer healing and in diabetic foot ulcer trials. In a two arm clinical study a 40% reduction of wound size compared to the control wound was observed in 6 weeks when used at 0.5 μg/cm2. However, in a 3 arm clinical study when 2.5 μg/cm2 was tested for comparison against standard XEROFORM® dressing, the results were not very encouraging.
4) Epidermal growth Factor (EGF) produced by platelets and macrophages is a mitogen for epithelial cells. This growth factor was first tested in burn patients and the initial results were promising. However, when tested in volunteers there was no difference between growth factor treatments and placebo. This could be due to the fact that EGF is not good for migration of keratinocytes, but is a good mitotic agent.
5) Keratinocyte Growth Factor-2 (KGF-2) was tested for its ability to increase ephithelialization. By day 6 the interstices were closed. KGF-2 promotes re-epithelialization in young and old animals suggesting indirect mechanisms for neo-granulation tissue formation. Xia Y. D., et al., J. Pathol. (1999) 188: 431–438. There is increased resistance to mechanical stress of healed wounds, hence KGF-2 may be useful for the treatment of surgical wounds. Jiminez, P. A. & Rampy, M. A., (1999) J. Surg. Res. 81: 238–242.
6) Connective tissue growth factor (CTGF) is a secreted, mitogenic, chemotactic and cell matrix inducing factor encoded by an immediate early growth responsive gene. Involvement of CTGF in human atherosclerosis and fibrotic disorders suggests a role in tissue regeneration like wound repair, but also in aberrant deposition of extracellular matrix. In fact, anti-CTFG antibodies have been used to block the fibrotic cascade.
Studies on the kinetics of action of various growth factors demonstrated that some growth factors such as granulocyte-monocyte colony stimulating factor (GMCSF) and bovine FGF acted sequentially. It was hypothesized that a combination of growth factors would be better than a single growth factor treatment. However, in animal models, a combination of these two factors actually slowed the regenerative process and healing never achieved 100%. Hence, sequential delivery of these factors was attempted: GMCSF was administered first followed by FGF delivery 25 days later. In a single study, no improvement over control could be demonstrated.
In yet another study combining TGF-β, bFGF (basic FGF) and CTGF it was found that TGF-β1, TGF-β2 or TGF-β3 caused skin fibrosis after 3 days of continuous injection but the change was transient and disappeared after 7 days of continuous injection. In contrast, irreversible fibrosis was observed upon simultaneous injection of TGF-β and bFGF or TGF-β and CTGF, or TGF-β injection for the first 3 days followed by bFGF or CTGF injection for the next 4 days. These observations suggest that TGF-β1 induces skin fibrosis and bFGF or CTGF maintains it in various skin fibrotic disorders.
Another way of obtaining growth factor mixtures considered the use of platelet releasate which contains a collection of growth factors released from platelets derived from blood. The advantages of this material are that it is autologous or homologous, and is readily available and presumably contains the required factors in the proper ratio. To date, although some improvement in the healing process was observed initially, by 24 weeks there was no difference between growth factor and placebo treatments.
It is thus apparent that although several polypeptide growth factors have shown significant biological activity in pre-clinical wound repair models, the only growth factor that has proven to be effective in the clinic is the human recombinant PDGF-BB. This may be due to poor delivery, drug instability or the inability of a single factor to orchestrate the complex process of wound healing. An effective treatment should address issues such as angiogenesis, efficient collagen deposition and proper epithelialization to close the wound.